This invention relates to a method, application of the method and a layer structure for determination of a substance.
Chemical sensors for the determination of chemical substances (so-called analytes) in samples or specimens comprise a whole range of thermodynamically reversible or regenerable measuring probes. Such probes must include at least one recognizing element, at least one transducer element, and, ideally, at least one calculation/display element. The recognizing element usually is implanted in a layer structure; the analyte is selectively isolated from other substances in the sample/specimen and extracted into the layer. Recognition molecules are also referred to as host molecules and are capable of selectively recognizing the analyte, which may be referred to as guest molecule. The process of recognition, the interaction between host and guest molecule, leads to a quantifiable signal which is obtained in a transducing process. Biosensors represent a subgroup of chemical sensors in which the recognizing element is a biologically active substance, such as enzymes, peptides, receptors, antibodies, cell organelles, etc.
Chemical sensors and biosensors feature a growing development potential and are of increasing importance in analytics (analyses in the fields of environmental and food technology, medicine and biotechnology). The characteristics of such sensors are:
inexpensive and rapid analyses, PA1 continuous measuring directly in the specimen, PA1 mobility of probes and measuring systems, PA1 use in isolated locations and high-risk sites, PA1 selective use of specifically tailored systems. PA1 ion-selective electrodes PA1 potentiometric electrodes PA1 ion-selective optodes PA1 optodes PA1 amperometric, enzymatic electrodes PA1 biosensors, immunosensors
2. Description of the Prior Art
2.1 Recognition molecules
Chemical recognition molecules as such are synthetic substances that are specifically designed for this purpose (W. Simon, U. E. Spichiger, Development and Application of Ion-Selective Electrodes, International Laboratory, September 1991, 35-43). In biosensors, on the other hand, molecules are used which are already known from biological processes, or are produced in biological systems. Some of them are enzymes, synthetic and gene-manipulated enzymes; antibodies, fragments of antibodies, synthetic and gene-manipulated antibodies; receptors and their hybrids; peptides, carbohydrates, lipids and their hybrids.
2.2 Design of sensors
As is known recognition molecules are implemented in the chemical probe in two different ways:
2.2.1 by immobilization on the surface of the supporting medium (electrostatic, covalent, and adhesive interactions, etc.); they may be brought into contact with both nonpolar and polar phases (Hall, G. F., Turner, A. P. F., J. Cell. Biochem., Suppl. 14B (1990) 345; Hall, G. F. et al., Anal. Chim. Acta 213 (1988) 113-119).
2.2.2 by incoporation into polar or nonpolar bulk membranes, so that molecular recognition takes place in or next to the boundary face rather than on the surface of the membrane. Relevant examples have been described in various publications (Seiler, K., Simon, W., Anal. Chim. Acta, 266 (1992) 73-87; patent publications EP-A-0153641, JP-A-2082152).
In the transducing process the step of chemical recognition is converted into a physical measurement variable. In the instance of optodes (optical sensors) the measurement variable is an optical signal, in the instance of electrodes (electro-chemical sensors) an electrical signal.
2.3 Existing sensors
Chemical sensors and biosensors are used for quantitative analysis of various analytes, such as
2.3.1 for ions and charged molecules
2.3.2 for uncharged, charged, or neutral molecules
2.4 Problems and drawbacks of existing sensor systems
As regards sensors in which the recognition molecule is immobilized on the surface (cf. 2.2.1) and exposed to the sample, the environment of the recognition process is poorly defined. As a rule the quality obtained in such instances does not meet the demands that are made on a reliable sensor. Typical deficiencies concern the life of the sensor, its invariance to pH, proteins, general nonspecific influences exerted by the matrix of the specimen or sample and ionic strength. Nonspecific interactions occur with the background of the sample/specimen. Regeneration of the recognition layer leads to the loss of expensive compounds, such as antigens and antibodies.
As regards sensors in which the recognizing molecule is implanted in a layer structure (cf. 2.2.2), the above drawbacks are not encountered. In this type of biosensors, however, the transducer layer usually is separated from the recognition layer. As a consequence, part of the signal is lost by diffusion; response time increases and the signal yield is reduced, as is described by Stamm for the urea optode (C. Stamm et al., Anal. Chim. Acta, 282 (1993), 229-237).
It is further known that biomolecules are solubilized in organic solvents by means of reverse micelles while retaining their biological activity (Luisi, P. L. et al., Methods in Enzymology, Vol. 136 (1987) 188-216, Acad. Press Inc.; Martinek, K. et al., Eur. J. Biochem. 155 (1986) 453-468). The three-component systems described comprise a tenside, and a polar hydrophilic, and a nonpolar lipophilic solvent; they are characterized by a parameter W.sub.o representing the ratio of water as polar solvent to tenside. The functionality of the enzymes solubilized therein was examined in dependence on the water content (Han, D. et al., Biocatalysis, Vol.4 (1990), 153-161).